Emetine Derivatives, Prodrugs Containing Same, And Methods Of Treating Conditions Using Same

ABSTRACT

Compounds are provided herein which are emetine derivatives that can be used as prodrugs which selectively undergo activation to release emetine in specific cellular conditions. In one aspect, a blocking group is incorporated onto the emetine molecule by the derivization of the N2′-position with moieties that can be selectively removed by hydrolysis in the cancer/tumor microenvironment. Such compounds are less cytotoxic than emetine and are substantially inactive in non-cancerous cells. In one aspect, the compounds described herein can be used for the treatment of metastatic and non-metastatic cancers, including, for example, breast cancer, prostate cancer, lung cancer, and leukemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2012/038655, filed May 18,2012, which claims the benefit of U.S. Provisional Application No.61/488,601, filed May 20, 2011, which are incorporated herein byreference in their entirety.

FIELD

The disclosure relates to compounds and prodrugs useful for treating acondition or disease, in particular a condition or disease that inducesa local decrease in pH or local over expression of an enzyme.

BACKGROUND

It was estimated by the National Cancer Institute (NCI) that 1,529,560new cases of cancer of all types would occur in 2010 and 569,490 deathswere statistically projected for the same year in the United States.Most solid tumors are uniformly fatal once they have disseminated beyondtheir tissue of origin. The efficacy of current cancer chemotherapy islimited by systemic toxicity and lack of tumor selectivity, resulting ina variety of side effects. Thus, there is a need to developcancer-specific agents for the treatment of both metastatic andnon-metastatic cancers.

Emetine is a natural product alkaloid found in the root of Psychotriaipecacuanha. Emetine has been shown to possess anti-cancer activitiesvia what are believed to be a variety of mechanisms of action. Emetineis an inhibitor of mitochondrial and ribosomal protein synthesis andalso interferes with the synthesis and activities of DNA and RNA.Emetine has a very significant anti-cancer potency and itschemotherapeutic action was evaluated up to Phase II clinical trials onseveral solid tumors about four decades ago. However, it was found thatemetine has a very narrow therapeutic index and can cause side effectslike muscle fatigue and cardiac toxicity.

It has been reported that N-(2-hydroxypropyl)-methacrylamide (HPMA)copolymer conjugates containing emetine were 60 times less toxic thanfree emetine in B16F 10 melanoma cells and 225 times less toxic in L1210leukemia cells. It was also reported that the prodrug positivelyaffected the survival of animals with L1210 tumors. However, the prodrugpolymer was found to only contain about 8% (wt/wt) of bound emetine anddid not significantly affect the rate of tumor growth. These findingsdiscouraged further development of emetine into a clinically usefulanticancer drug.

SUMMARY

Compounds are provided herein which are emetine derivatives that can beused as emetine prodrugs which selectively undergo activation to releaseemetine in specific cellular conditions. In one aspect, a blocking groupis incorporated onto the emetine molecule by the derivatization of theN2′ position with moieties that can be selectively removed byhydrolysis. Protonation and hydrolysis begins a cascade of one or morereactions in which the bond between the blocking group and the nitrogenat the N2′-position is cleaved with hydrogen replacing the blockinggroup in the cancer/tumor microenvironment. This exposes the cancercells to emetine which heretofore was too toxic to be useful for cancertreatment. Such emetine derivatives with the blocking group are lesscytotoxic than emetine and are substantially inactive in non-cancerouscells. In one aspect, the compounds described herein can be used for thetreatment of metastatic and non-metastatic cancers, including, forexample, breast cancer, leukemia, lung cancer, and prostate cancer.

While not intending to be limited by theory, it is presently believedthat the protein synthesis inhibitory activity of emetine andconsequently its anticancer activity are dependent on the availabilityof the N2′-position as a secondary amine. It was found that compoundscarrying a substituent at the N2′-position show reduced toxicities incells and tissues at low concentrations and thus do not show the samecardiotoxicity as emetine. Further, the removal of the substituent incells or tissues will release pure emetine as a potent antitumor agent.In one aspect, selective pH-dependent or enzymatic removal of thissubstituent in the cancer environment avoids general systemic toxicityand cardiotoxicity of these compounds and ensures that the prodrugs andassociated toxicity are targeted to cancer cells.

In one aspect, the compounds provided herein selectively undergoactivation to release emetine in acidic pH, such as in the slightlyacidic environment of cancer cells. In this aspect, the compoundprovided herein includes an acid labile functional group linked to theN2′-position of emetine, such as by covalent bond. The covalent bondlinking the functional group to the N2′-position of emetine is labile inelevated concentrations of hydrogen ions, such that the covalent bond ishydrolyzed at a pH less than about 7.0, in another aspect at a pH ofless than about 6.9, and in another aspect at a pH of less than about6.8.

In another aspect, compounds which selectively undergo activation torelease emetine by enzymatic hydrolysis are provided. Such compounds areuseful as prodrugs. In this aspect, enzyme-activated compounds whichhave emetine coupled to a peptide or other pharmacophore via aself-cleaving linker are provided. The compound is then activated byenzymes preferentially expressed by and/or specific to the cancer cellor immediate environment of the cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a graph showing the viability of DU145 cells as percentof vehicle on day 5 versus concentration of emetine and Compound 2.

FIG. 2 includes a graph showing the viability of PC3 cells as percent ofvehicle on day 5 versus concentration of emetine and Compound 2.

FIG. 3 includes a chart showing reduction of average growth medium overseven days due to the metabolism of PC3 prostate cancer lines.

FIG. 4 includes a chart comparing growth of PC3 cell lines in growthmedium of pH 6.8-7.0 and pH 7.4.

DETAILED DESCRIPTION

The compounds provided herein include prodrug forms of therapeuticagents with a hydrolyzable group on the N2′-position of emetine. Thecompounds carry a substituent at the N2′-position. The compoundsprovided herein selectively undergo activation to release emetine inspecific cellular environments by selective pH-dependent or enzymaticremoval of the substituent in a desired environment. Emetine has thefollowing structure:

The compounds described herein are useful as prodrugs for the treatmentof a variety of medical conditions, including, for example, metastaticand non-metastatic cancers. In one aspect, the compounds are useful asprodrugs for the treatment of breast cancer, leukemia, lung cancer, andprostate cancer.

The compounds described herein can be administered to a subject insubstantially stable, inactive form and will remain in substantiallystable, inactive form until either hydrolyzed in the acidic environmentof the cancer cell or when activated by an enzyme specific to the cancercell or immediate environment of the cancer cell. By either approach,once in or around the cancer cell, the prodrug is activated byhydrolysis, resulting in the release of free emetine, which is effectiveto kill the cancer cell and/or prevent its proliferation. While theprodrug may also be activated in non-cancerous cells and possibly harmthe non-cancerous cells, the amount of activation in non-cancerous cellsis low and toxicity to those cells is minimized.

By one approach, the compounds provided herein include an acid labilefunctional group “M” linked to the N2′-position of emetine as shownbelow in formula (I).

By one approach, the bond between M and the nitrogen at the N2′-positionis cleaved at acidic pH, such as in the slightly acidic environment ofcancer cells as compared to the higher pH (e.g., about 7.3) of blood andnormal cells (e.g., non-cancerous and/or non-infected cells). In oneaspect, M is selectively removed at a pH of less than about 7.0, inanother aspect less than about pH 6.9, and in another aspect at a pH ofless than about 6.8, and the compounds are substantially stable and nothydrolyzed at the higher pH of blood and normal cells. Such selectiveremoval of this group avoids general systemic toxicity andcardiotoxicity of the compounds and ensures that the toxicity of thecompounds is substantially targeted to cancer cells. The hydrolysis ratewithin the cancer cells is high enough to liberate a sufficient quantityof emetine in the cancer cells to be pharmaceutically useful.

In one aspect, Group M is selected from the group consisting of:

wherein:

X₁═H, —NO₂, —CO₂X₂, —OX₃, halogen, or C1-C4 alkyl,X₂═C1-C4 alkyl or H,X₃═C1-C4 alkyl or H,

Q=OH, V⁺O⁻,

V=metal ion,Y═C1-C6 alkyl or H,Z═H, C1-C4 alkyl, or halogen,p=0 to 8, andt=1 to 4.

Other configurations and substituents of Group M may also be provided solong as the group is electron-donating such that the bond between GroupM and the N2′-position of emetine is hydrolyzed at a pH of less thanabout 7.0, in another aspect at a pH of less than about 6.9, and inanother at a pH of less than about 6.8, to release emetine inpharmaceutically useful amounts.

In another aspect, Group M has the general formula:

where V is a metal ion.

In yet another aspect, Group M has the general formula:

where R₈ is selected from the group consisting of:

wherein:

L=H₂N— or H₃CO—

m=1-3.n=0-1,p=0 to 8,X₁═H, —NO₂, —CO₂X₂, —OX₃, halogen, or C1-C4 alkyl,X₂═C1-C4 alkyl or H, andX₃═C1-C4 alkyl or H.

Where V is a metal ion in the compounds described herein, the metal ionmay be, for example, sodium or potassium. Other metal ions may also beused, if desired. In one aspect, the metal ion has a valence of 1.

Some of the compounds described herein can comprise one or moreasymmetric centers, and thus can exist in various isomeric forms, suchas stereoisomers and/or diastereomers. Compounds may be in the form ofan individual enantiomer, diastereomer or geometric isomer or may be inthe form of a mixture of stereoisomers. In some approaches, thecompounds are enantiopure compounds. In other approaches, mixtures ofstereoisomers or diastereomers are provided.

Some of the compounds provided herein may have one or more double bondsthat can exist as either the Z or E isomer, unless otherwise indicated.In one approach, the compounds are provided as individual isomerssubstantially free of other isomers and alternatively, as mixtures ofvarious isomers, e.g., racemic mixtures of stereoisomers. In anotherapproach, pharmaceutically acceptable derivatives of these compounds areprovided.

It was found that carbamates, thiocarbamates, and dithiocarbamates areoften more enzymatically stable than the corresponding amides but aremore easily hydrolyzed than amides. The pH of hydrolysis—and thus theease of hydrolysis—can be tunable depending in the group attached to theN2′-position of emetine. Incorporation of electron-donating groups inR₁-R₈ above facilitates the acidic cleavage in the lysosome of the cell.For example, acid-catalyzed hydrolysis of a carbamate to carbamic acidfollowed by spontaneous elimination of carbon dioxide provides emetinein an acidic environment as shown in Scheme 1 below.

Enzyme Activated Emetine Prodrugs

By another approach, prodrugs provided herein are preferentiallyactivated by one or more enzymes overproduced or selectively produced bycancer cells. The prodrugs are activated by removal of a substituent atthe N2′-position of emetine. Removal of the substituent may require oneor more hydrolysis and/or self-cleavage steps to provide free emetine.In some approaches, the prodrugs include a peptide sequence of asubstituent at the N2′-position of emetine that is hydrolyzed by anenzyme that is predominantly present in cancer cells. Hydrolysisprovides an intermediate compound that self-cleaves to provide freeemetine.

By one approach, a compound is provided having formula (I):

where M is not hydrogen and comprises a substituent effective to behydrolyzed by an enzyme preferentially expressed by cancer cells. Insome aspects, M comprises one of

where X is a protecting group or one or more amino acids. For example,the protecting group could include 9-fluorenylmethyloxycarbonyl(“Fmoc”), tert-butyloxycarbonyl (“t-Boc”), acetyl, and morpholinogroups. Other protecting groups as are known in the art may also beused, if desired.

In one aspect, the tumor stromal protease fibroblast-activation protein(FAP), a serine protease previously demonstrated to be expressed bystroma of more than 90% of tested human cancers, can be utilized forselective activation of a prodrug in accordance with the presentdisclosure. FAP is also expressed by mouse stroma within humanxenografts. The compounds provided herein include prodrugs containingvarying peptide sequences that are hydrolyzable by FAP. The peptidesequences are provided in a group at the N2′-position of emetine. FAPhydrolysis leads to a self-cleaving para-aminobenzylcarbamateintermediate which, upon electronic rearrangement of the linker(self-cleavage), releases free emetine. The release of emetine by thisapproach is demonstrated in Scheme 2 below.

In another aspect, a prodrug is provided having a peptide with the aminoacid sequence His-Ser-Ser-Lys-Leu-Gln (HSSKLQ) (SEQ ID NO.: 1), that isselectively and efficiently hydrolyzed by Prostate Specific Antigen(PSA). PSA levels in men with prostate cancer can exceed 1000 ng/mL. PSAis enzymatically made inactive by binding to the major proteaseinhibitors α1-antichymotrypsin and α2-macroglobulin, which are generallyfound at a 104- to 105-fold molar excess in blood serum. However, PSA isfound in its active form in the immediate extra-tumoral environment.Thus, PSA can be employed for selective prodrug activation in theimmediate extra-tumoral environment. Therefore, emetine prodrugscleavable by PSA using a peptide comprising the sequence HSSKLQ (SEQ IDNO.: 1) can be used as prostate cancer chemotherapy. A PSA-activatedemetine prodrug is shown in Scheme 3 below.

In another aspect, a composition comprising a PABA-linked PSA cleavableemetine prodrug is provided. The prodrug is hydrolyzable by ProstateSpecific Antigen (PSA) in vivo and in vitro to provide an intermediatethat self-cleaves to provide free emetine. A PABA-linked PSA-activatedemetine prodrug is shown in Scheme 4 below.

In another aspect, a composition comprising an ethylenediamine-linked(EDA-linked) PSA cleavable emetine prodrug is provided. The prodrug ishydrolyzable by PSA in vivo and in vitro to provide an intermediate thatself-cleaves to provide free emetine. An EDA-linked PSA-activatedemetine prodrug is shown in Scheme 5 below.

In yet another aspect, a PABA-linked emetine prodrug that is cleavableby Fibroblast Activation Protein (FAP) is provided. The prodrug isproviding having an amino acid sequence Asp-Arg-Gly-Glu-Thr-Gly-Pro (SEQID NO.: 2) that is selectively and efficiently hydrolyzed by FAP invitro and in vivo. FAP is a 95 kDa glycoprotein expressed by tumorstromal fibroblasts in most cancers. Therefore, FAP can be employed forselective activation of emetine prodrugs cleavable by FAP using apeptide comprising the sequence Asp-Arg-Gly-Glu-Thr-Gly-Pro (SEQ ID NO.:2), and can be used for cancer treatment.

A PABA-linked FAP-activated emetine prodrug is shown in Scheme 6 below.

In yet another aspect, an EDA-linked emetine prodrug that is activatableby FAP as generally described above is also provided. An EDA-linkedFAP-activated emetine prodrug is shown in Scheme 7 below.

In yet another aspect, a pH low insertion peptide (pHLIP)-emetineconjugate is provided. In this aspect, a water-soluble membrane peptidethat interacts weakly with the cell membrane at neutral pH is linked atits c-terminus to emetine at the N2′-position. At slightly acidic pH(i.e., less than about pH 7.0), pHLIP inserts into the cell membrane andreleases emetine into the cytoplasm. In this approach, emetine isconnected to a linker that is conjugated to the inserting C terminus ofpHLIP through a disulfide bond that will be cleaved inside the cells,thereby releasing emetine. pHLIP has the following amino acid sequence:AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG (SEQ ID NO.: 3). A pHLIP-emetineconjugate is provided below. The conjugate is suitable for the targeteddelivery of emetine into the acidic cancer microenvironment.

In one aspect, a pharmaceutical composition is provided which comprisesa prodrug described herein. The pharmaceutical composition may furthercomprise a pharmaceutically acceptable adjuvant or vehicle, if desired.Pharmaceutically acceptable adjuvants or vehicles include, for example,Exemplary pharmaceutically-acceptable carriers include saline, bufferedsaline, isotonic saline, Ringer's solution, dextrose, sterile water,deionized water, glycerol, ethanol, 5% dextrose in water, propyleneglycol and combinations thereof.

The compositions described herein may be administered to a subject by avariety of modes of administration. As such, the formulation as well asthe concentration of the composition may vary. In one aspect, thecompositions may be applied directly to target tissues or organs, or tosurrounding fluid or tissue. By one approach, the composition may beadministered to a subject via a variety of routes, including, forexample, parenterally, particularly intravenously. In one aspect,administration to the desired location may be done by catheter, infusionpump, or stent. Liquid formulations can be prepared, such as, forexample, in the form of a solution or suspension in a non-toxic,parenterally-acceptable solvent or diluent. In another aspect, theformulation may be a powder or lyophilate that is reconstituted with asolvent prior to use. In yet another aspect, the formulation may be inthe form of an emulsion or liquid concentrate for dilution prior toadministration. Exemplary pharmaceutically-acceptable carriers includesaline, buffered saline, isotonic saline, Ringer's solution, dextrose,sterile water, deionized water, glycerol, ethanol, 5% dextrose in water,and combinations thereof.

A method for the therapeutic treatment of a medical condition is alsoprovided. The method includes administering to a subject atherapeutically effective amount of a prodrug composition providedherein. Human patients are typically the recipients of the compositionsprovided herein, although veterinary usage is also contemplated. By oneapproach, the medical condition being treated is metastatic ornon-metastatic cancer, including, for example, breast cancer, leukemia,lung cancer, and prostate cancer. In a more particular aspect, themedical condition is prostate cancer.

In another aspect, a method of delivering emetine to a subjectcomprising administering to the subject an effective amount of a prodrugcompound according to any of the aspects described herein.

In yet another aspect, a method for decreasing the toxicity of emetineis provided, the method comprising synthesizing a prodrug comprising acompound according to any of the aspects described herein.

The following examples are provided to illustrate certain aspects of thedisclosure but should not be construed as limiting the scope of thedisclosure. Unless specified otherwise, all percentages are by weight.

EXAMPLES General Methods Example 1 Synthesis of Dithiocarbamate EsterDerivatives of Emetine

A solution of NaOH or KOH (3 molar equiv.) in water (1.00 ml or about 5%or less of volume of ethanol) and ethanol (20.00 mL) was added to asolution of emetine dihydrochloride hydrate (1.11 g, 2.00 mmol) inethanol (10.0 ml) at −8° C. This was stirred at this temperature for 15minutes after which CS₂ (0.30 mL, 4.97 mmol, 2.5 molar equiv.) wasadded. The resulting mixture was stirred at −5 to 1° C. for 2 hours andat room temperature for 30 minutes. The solvent was removed in vacuo andthe residue triturated with acetonitrile and then filtered. The filtratewas evaporated to dryness and the residue dissolved in ethyl acetate (3mL). To this was added diethyl ether which afforded the precipitation of“Compound 1” or “Compound 2,” as shown in Scheme 8 below, as a whitesolid (82.0% yield).

The dithiocarbamate ester analogs can then be synthesized from the saltsof compounds 1 or 2 above as depicted in Scheme 9 below. A Radley'sCarousel reaction station with twelve reaction tubes (24 mm×150 mm) wasused. Compound 2 (200 mg, 0.35 mmol) was weighed into each of the twelvereaction tubes, and acetonitrile (15 mL) was added to each tube todissolve the salt. Each of the twelve alkylating agents (0.27 mmol),labeled 3a-3l below, was dissolved in acetonitrile (5 mL). Each reactiontube containing the dissolved salt was then charged with one of thetwelve alkylating agents. The reaction tubes were capped and stirred for24 hours. The mixture in each reaction tube was then transferred to a100-mL round bottom flask and the solvent was evaporated in vacuo. Theresidue obtained after evaporation of the solvent was triturated withwater (20 mL) to dissolve any inorganic substances and then filteredunder suction. Each crude product was air-dried and then purified byflash chromatography on silica gel using EtOAc: MeOH (10:1) as eluent.

Alkylating agents (RX) useful herein include, for example, thefollowing:

The following dithiocarbamate ester analogs of emetine (4a-4l) wereobtained:

Example 2 Synthesis of Carbamate Derivatives of Emetine

Emetine dihydrochloride (1.0 molar equiv.) was added at room temperatureto a stirred solution of dimethyl amino pyridine (DMAP) or triethylamine (4.0 molar equiv.) in chloroform. After ten minutes, theappropriate chloroformate (4 molar equiv.) was added. The reactionmixture was then stirred at room temperature for 12-24 hours. Thesolvent was removed in vacuo and the residue was dissolved in CH₂Cl₂ (25mL) and washed with water (2×20 mL) and brine (1×20 mL). It was thendried over anhydrous MgSO₄ and solvent was evaporated under reducedpressure. The crude product was purified by column chromatography onsilica gel eluting with 100% EtOAc to EtOAc:MeOH 7.5:2.5 to give thedesired product. The reaction scheme for synthesis of carbamate esteranalogs of emetine is shown in Scheme 10 below.

The following carbamate ester analogs of emetine (5a-5c) were obtained:

Example 3 Synthesis of Sulfonamide Derivatives of Emetine

To a stirred solution of dimethyl amino pyridine (DMAP) (4 molar equiv).in CH₂Cl₂ (20 mL) was added emetine dihydrochloride (1 molar equiv) atroom temperature. After 15-20 minutes, the appropriate sulfonyl chloride(2.5 molar equiv) solution in CH₂Cl₂ was added. The reaction mixture wasthen stirred at room temperature for 12-16 h. The solvent was removed invacuo and water (25 mL) was added to the residue to dissolve all thewater soluble impurities. Then the crude product was isolated byextraction into CH₂Cl₂ (3×25 mL). The combined organic phase was thenwashed with brine (2×20 mL), dried over anhydrous MgSO₄ and filtered.Solvent was evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel using gradient elutionstarting with 100% CH₂Cl₂ to remove the least retained impurities andgradually varying this to CH₂Cl₂: MeOH eluent mixture with optimum MeOHcomponents varying from 5 to 20% depending on the specific analog(8a-8f) to afford the desired product. The reaction is illustrated belowin Scheme 11.

The following sulfonamide analogs of emetine (8a-8f) were obtained:

Example 4 Synthesis of Thiorurea Derivatives of Emetine

The general reaction scheme for synthesis of thiourea analogs of emetineis shown in Scheme 12 below.

Isothiocyanates were first synthesized according to JOC: 72, 3969-3971(2007), which is incorporated herein by reference, with slightmodification. To a solution of appropriate amine (16.0 mmol, 1 molarequiv.) in THF (15 mL) at 0° C. was added triethylamine (10.0 mL). Theresultant mixture was kept stirring while CS₂ (34.0 mmol, 2 molarequiv.) was added dropwise over about thirty minutes at 0° C. Themixture was allowed to stir at this temperature for fifteen minutesafter which it was stirred at room temperature for one hour. Thereaction mixture was then cooled to 0° C. again while stirringcontinued, and a solution of tosyl chloride (20.8 mmol, 1.3 molar equiv)in THF was added gently. The reaction mixture was allowed to warm up toroom temperature, stirred for an additional hour at room temperature andthen 20 mL 1N HCl was added while stirring continued. This was followedby 25 mL diethyl ether and the reaction was stirred for another fiveminutes. The aqueous layer was separated and then back extracted withdiethyl ether (2×20 mL). The combined organic layers were dried overNa₂SO₄, solvent evaporated in vacuo, and the crude product was purifiedby column chromatography eluting with hexanes over silica gel to givepure isothiocyanate which was used in the next synthesis step.

The second step is synthesis of thiourea analogs of emetine fromisothiocyanate. To a stirred solution of pyridine or triethylamine (0.5mL) in CH₂Cl₂ (15 mL) at room temperature was added emetinedihydrochloride (200 mg, 0.36 mmol, 1 molar equiv.). After all theemetine was completely dissolved, the appropriate isothiocyanate, 10a-n,(0.72 mmol, 2 molar equiv.) was added. The reaction mixture was stirredat room temperature for 8 to 16 hours. CH₂Cl₂ (15 mL) was then added tothe mixture which was subsequently washed with water (2×25 mL) and brine(1×25 mL). The organic layer was dried over MgSO₄ and the solventremoved in vacuo. The crude product was purified either by precipitationin a diethyl ether/hexane mixture or by column chromatography usingCH₂Cl₂/MeOH mixture in appropriate ratio as eluent. Compounds 11a-d and11f-h were purified by precipitation in a 1:1 and 3:7 mixture(respectively) of diethyl ether and hexanes; whereas 11 e, and 11i-nwere all purified by column chromatography over silica gel usinggradient elution. Elution with 100% CH₂Cl₂ separated the nonpolarimpurities in all of them while 10% MeOH in CH₂Cl₂ was the best eluentto afford pure 11 e, 11i and 11j. However, 5% MeOH in CH₂Cl₂ was theoptimum eluent system for obtaining pure form of 11k-n.

The following thiourea analogs of emetine (11a-11n) were obtained:

Example 5 Synthesis of Urea Analogs of Emetine

The synthesis of urea analogs was done in two stages as shown in Scheme13 below.

First, isocyanates were synthesized from an appropriate amine, using atwo-step but one pot reaction. The procedure was reported in JOC 1996,61, 3883-3884, which is incorporated herein by reference. Theisocyanates were then reacted with emetine to make the urea analogs.

A solution of trichloromethyl chloroformate (5.741 mmol, 1.5 molarequiv) in CH₂Cl₂ (15 mL) at 0° C. was set stirring. To this was addeddropwisely a solution containing a mixture of an appropriate amine, 9e-gand 9k, (3.827 mmol, 1 molar equiv) and1,8-bis(dimethylamino)-naphthalene or DMAP (7.654 mmol, 2 molar equiv)over about 5 to 10 min. Thereafter, the ice bath was removed and thereaction mixture was allowed to warm up to room temperature and thenstirred for another 45 min. Solvent and all volatiles were evaporated invacuo and fresh CH₂Cl₂ 30 mL was added followed by 1N HCl solution (20mL); this was stirred for about 3-5 min. The organic layer was separatedand then washed with 1 N HCl (3×15 mL) and 1 N NaOH (1×15 mL). It wasdried over Na₂SO₄ and solvent was evaporated in vacuo to give therespective isocyanates, 12e-g and 12k, that were used without furtherpurification.

To a solution of a given isocyanate, 12e-g and 12k (0.72 mmol, 2 molarequiv.) in CH₂Cl₂ (10 mL), was added a solution of emetinedihydrochloride (200 mg, 0.36 mmol, 1 molar equiv) and DMAP (1.44 mmol,4 molar equiv.) mixture in CH₂Cl₂ (15 mL) at room temperature. Thereaction mixture was stirred overnight at room temperature. 10 mL CH₂Cl₂was then added to the mixture which was then washed with water (2×25 mL)and brine (1×25 mL). The organic layer was then dried over MgSO₄followed by solvent removal in vacuo. All the products were purified bycolumn chromatography using gradient elution beginning with 100% EtOActo an eluent containing 10% MeOH in EtOAc to afford pure 13e-g and 13k.

The following urea analogs of emetine (13e-g and 13k) were obtained.

Example 6 Synthesis of Amide Derivatives of Emetine

A solution of emetine dihydrochloride (1 molar equiv) in a chloroform(10 mL to 500 mg emetine) and triethylamine (2 mL) was stirred for about5 minutes. To this was added a solution of the appropriate anhydride14a-e (4 molar equiv.) in chloroform. This reaction mixture was stirredfor 8 to 20 hours at room temperature. MALDI-MS was utilized toqualitatively determine the formation of the desired product in thereaction mixture. All volatiles and solvent was evaporated in vacuo fromthe reaction mixture to give the crude products of the amide analogs15a-e. Purification methods vary slightly from one analog to another andthese are specified below. The general reaction scheme is shown below.

For compounds 15a, b, and e, the crude product was dissolved in about 40mL of chloroform. This was washed with water (2×20 mL) and the organicphase later washed with an aqueous solution containing 5% 2N NaOH indistilled water (1×20 mL) to remove all the triethyl amine and thenobtain sodium salt at the carboxylate end of these amides. The organicphase was later washed with brine (15 mL×2) and then dried with MgSO₄.Solvent was completely evaporated in vacuo at a temperature not greaterthan 45° C. The crude product obtained was dissolved in as little ethylacetate as possible (about 1.0 mL of ethyl acetate for a reaction of 500mg of emetine.2HCl). Hexanes (about 15-20 mL) were then added to thismixture in order to precipitate the pure products which were filteredunder vacuum and then washed with a lot of hexanes. The solid productsobtained (white to pale yellow) were then dried in vacuum over at 70° C.for 4 hours in order to remove all traces of solvent impurities.

For compounds 15c and d, the crude product was dissolved in 50 mL ofchloroform, and then washed in water (1×20 mL). This must be done verygently and carefully to avoid emulsion formation. Then it was washedwith a solution of 5% 2 N aqueous NaOH in brine (2×20 mL). The organicphase was later washed in 100% brine (1×20 mL), and then dried overMgSO₄. Solvent was evaporated in vacuo at a temperature not greater than45° C. The crude product obtained was dissolved in as little ethylacetate as possible (about 1.0 mL of ethyl acetate for a reaction of 500mg of emetine.2HCl). Hexanes (about 15-20 mL) was then added to thismixture in order to precipitate the pure products which were filteredunder vacuum and then washed with a lot of hexanes. The solid productsobtained (white to pale yellow) were then dried in vacuum over at 70° C.for 4 hours in order to remove all traces of solvent impurities.

The following products were obtained:

Example 7 Synthesis of Carbamate and Thiocarbamate Derivatives ofEmetine

Carbamate and thiocarbamate derivatives can be synthesized fromchloroformates or chlorothioformates in high yield by employing theSchotten-Baumann procedure as outlined in Scheme 15 below.

Example 8 Synthesis of Dithiocarbamate Derivatives of Emetine

The synthesis of dithiocarbamate esters is accomplished by convertingemetine to the potassium dithiocarbamate salt followed bystraightforward S_(N)2 reaction of the salt with various benzyl halidederivatives as shown in Scheme 16.

Example 9 Cytotoxicity of Emetine and Compound 2

The cytotoxicity of emetine and Compound 2 (as produced above in Example1 and referred to in the figures as “ESA44”) was evaluated in DU145 andPC3 cells using 100% confluent cells. Cells were plated at 40,000 cellsper well and allowed to grow for 4 days until the pH of the medium was6.7. Compound 2 was added in RPMI, pH 7.0. The effect of the pH on thehydrolysis of Compound 2 to release emetine was evaluated. FIGS. 1 and 2are graphs showing the viability of DU145 and PC3 cells as percent ofvehicle on day 5 versus concentration of emetine and Compound 2. Theresults suggest that Compound 2 is hydrolyzed into emetine under moreacidic conditions.

The IC₅₀ values (in μM) are presented in Table 1 below.

TABLE 1 Fold at high Fold at low cell cell density and density andEmetine Compound 2 pH <7 pH >7.4 DU145 12.921 ± 2.39  11.192 ± 1.149~1.0 13.7 PC3  3.854 ± 0.149  7.463 ± 0.308 1.94 16.5

Example 10 Cytotoxicity of Emetine and Dithiocarbamate Salt

Studies were carried out on three prostate cancer cell lines: LNCaP,PC3, and DU145. The IC₅₀ of emetine after a 3 day exposure was 32.9 nMin LNCaP, 35.1 nM in PC3, and 37.9 nM in DU145, and after a seven dayexposure, the values were 31.6, 29.4, and 23.4 nM, respectively. Therewas not a significant difference in cytotoxicity of emetine between days3 and 7. However, for the dithiocarbamate salt (Compound 2), in vitrostudies revealed an IC₅₀ of 564.9 nM in LNCaP (about 17.2 fold reductioncompared to emetine), 442.0 nM in PC3 (about 12.6 fold) and 376.8 nM inDU145 (about 9.9 fold) after a 3 day exposure. A gradual increase inactivity was seen as exposure to the drug progressed to day 7 and IC₅₀values of 79.0, 87.1, and 79.3 nM respectively, was observed. Thisincrease is most likely due to the release of emetine from thedithiocarbamate salt over the time period. Further drug release studiesat pH 5 confirmed about 45% release of emetine from the dithiocarbamatesalt after 3-day incubation at this acidic pH maintained at 37° C.,whereas release of emetine from the dithiocarbamate salt was notobserved at physiological pH (e.g., about 7.4).

Example 11 Cytotoxicity of Various Compounds of Examples 1-5

Various compounds prepared according to Examples 1-5 were studied inLNCaP, PC3, and DU145 cells. The IC₅₀ was determined at 7 days and theresults are presented in Table 2 below.

TABLE 2 IC₅₀ in μM after 7 days COMPOUNDS LNCaP PC3 DU145 Emetine 0.0278± 0.00384  0.0268 ± 0.00228 0.0237 ± 0.00122 1  0.079 ± 0.00341  0.0871± 0.00499 0.0793 ± 0.00247 4b >10 6.562 ± 1.113 >10   4c 1.656 ± 0.5642.706 ± 0.192 2.467 ± 0.263 4d 2.77 ± 0.06 3.05 ± 0.07 4f  1.97 ± 0.0881.56 ± 0.29  1.98 ± 0.213 4g 1.698 ± 0.187 2.768 ± 0.146 2.795 ± 0.1514h 1.613 ± 0.066 3.027 ± 0.16    2.3 ± 0.067 4i 2.692 ± 0.145 2.449 ±0.162 4l 2.308 ± 0.174 4.855 ± 0.153 2.253 ± 0.084 5b 2.1622 ± 0.122  8b2.097 ± 0.284 4.012 ± 0.868 8e 2.263 ± 0.798 >10.0 >10.0 8a 6.241 ±0.098 8d 4.214 ± 0.435 6.619 ± 0.167 8c 4.576 ± 0.383 8.038 ± 0.06  8f6.075 ± 0.105 11a  0.484 ± 0.0247 11b 0.467 ± 0.025 11c 0.505 ± 0.015911d  0.339 ± 0.0327 0.443 ± 0.0383 11e 2.057 ± 0.438 6.916 ± 0.07111.535 ± 0.254 11g  1.59 ± 0.151 2.864 ± 0.0662  2.32 ± 0.091 11h 2.366 ±0.112 11f 5.002 ± 0.307  11j 2.627 ± 0.106 11i 5.271 ± 0.372 11k 1.313 ±0.166 6.915 ± 0.170  6.587 ± 0.0348 11l 1.700 ± 0.144 2.454 ± 0.323 11m1.006 ± 0.249 1.425 ± 0.336 11n >10   13e 2.115 ± 0.576

Example 12 Cytotoxicity of Compounds of Example 6

Various compounds prepared according to Example 6 were studied in DU145and PC3 cells. The IC₅₀ was determined at 3, 5, and 7 days. The resultsare presented in Tables 3 and 4 below.

TABLE 3 IC₅₀ in μM in DU145 COMPOUNDS Day 3 Day 5 Day 7 15a2 7.9611 ±0.3119 5.7299 ± 0.1819 4.2273 ± 0.4183 15b2 6.5257 ± 0.0852 5.9855 ±0.2385 5.2184 ± 0.0459 15c2 0.2477 ± 0.0418 0.09413 ± 0.00211 0.08454 ±0.00474 15d2 2.5764 ± 0.1905 2.0127 ± 0.0734  1.792 ± 0.1339 15e2 0.7331± 0.022  0.6408 ± 0.0543 0.8019 ± 0.0198

TABLE 4 IC₅₀ in μM in PC3 COMPOUNDS Day 3 Day 5 Day 7 15a2 8.3891 ±0.2083  7.0582 ± 0.01566 7.7083 ± 0.1507 15b2 7.5793 ± 0.1350 5.9855 ±0.2385 7.5141 ± 0.1677 15c2 0.5282 ± 0.0237  0.4239 ± 0.00785 0.4865 ±0.0688 15d2 3.0639 ± 0.1105  2.426 ± 0.1183 2.8886 ± 0.1233 15e2  2.495± 0.0722  1.818 ± 0.1613 2.149 ± 0.131

Example 13 Evaluation of pH-Responsiveness of Compounds by HPLC

Relative rates of hydrolysis of the compounds listed in Table 5 belowover a 48 hour period were evaluated using the percent emetine releasedfrom the acid catalyzed hydrolysis of compounds 1 to 6.

TABLE 5 pH Activated Emetine Analogs

1

2

3

4

5

6

The compounds were incubated in aqueous phosphate buffer at pH 5.5, 6.5and 7.4 at 37° C. over a 48 hour period. High performance liquidchromatography (HPLC) was employed to analyze the samples and quantifyhow much emetine was released. The data is summarized in Table 5 below.The compounds whose hydrolysis to emetine is shown in table 5 are all100% stable at physiological pH 7.4 up to a 5 day exposure. All thecompounds studied here were activated to emetine at pH 5.5 over a 48hour period, although to different extents. Percent hydrolysis toemetine was found to drop at the less acidic pH 6.5.

The rates of hydrolysis of the sodium salts 4-6 and the free acid 2 and3 are similar to that of maleic anhydride derivatives. Hence, amideanalogues synthesized from maleic anhydride and its derivatives in thisstudy showed great promise as seen particularly in compounds 2, 3 and 5which release about 50%, 80% and 50% emetine, respectively, within 48hours at pH 6.5. In addition, the free acid analogs 2 and 3 appear to bemore pH-responsive and sensitive than the corresponding sodiumcarboxylate salts 4 and 5, respectively. The pH responsiveness of thesodium dithiocarbamate salt 1 is higher at pH 5.5 than at 6.5 producingabout 94.5% hydrolysis within 48 hours at pH 5.5, but only 13.2% overthe same period at pH 6.5. The hydrolysis of 1 appears to be slower atpH 6.5 and it is therefore conceivable that there is an increase inamount of emetine released between day 3 and day 5 in the in vitrostudy, thereby showing that the compound likely has optimum activity byday 5.

TABLE 5 pH-responsiveness of emetine pro-drugs 1 to 6 in aqueoussolutions of pH 5.5 and 6.5; and stability in aqueous solutions of pH7.4 to 8.0 % Emetine* at % Emetine* at % Emetine* at pH 7.4 pH 5.5 pH6.5 to 8.0 over 72 hrs Compounds 0 hr 24 hrs 48 hrs 0 hr 24 hrs 48 hrs 0hrs 24 hrs 48 hrs 1 0.0% 55.9% 94.5% 0.0% 5.2% 13.2% 0.0% 0.0% 0.0% 20.0% 36.3 54.10 0.0% 35.5% 52.1% 0.0% 0.0% 0.0% 3 0.0% 78.1 93.4 0.0%76.8% 82.7% 0.0% 0.0% 0.0% 4 0.0% 19.2% 27.1% 0.0% 8.2% 17.1% 0.0% 0.0%0.0% 5 0.0% 61.5% 77.3% 0.0% 24.9% 49.2% 0.0% 0.0% 0.0% 6 0.0% 33.9%42.1% 0.0% 13.4% 21.1% 0.0% 0.0% 0.0% *% Emetine = Percent compositionof emetine in the mixture due to acid initiated hydrolysis at differenttime points after incubation at 37° C.

Example 14 In Vitro Cytotoxic Studies of Compounds 1 and 5 in PC3 CellLine Under Pre-Established Acidic Cancer Cell Culture Medium (pH<7.0)

Upon establishing the release of emetine from these compounds undermildly acidic condition, it was believed that the potency of thepH-responsive analogs could be increased if the cancer cell was adaptedto an acidic medium, so that the pH of the cell culture is already below7 at day 0. To investigate this, compounds 1 and 5, which are two of thethree most cytotoxic pH-responsive analogs, were selected. The range ofpH changes of the PC3 cell lines in the in vitro studies over a 7 dayperiod was established. The effects of the metabolism of PC3 cell lineson change in pH of growth medium without any drugs was investigated andthe results are presented in FIG. 3. This study showed that themetabolic activities of the PC3 cancer cells cause a fall in the pH ofthe growth medium from 7.4 on day 0 to 7.05 on day 3 and 6.66 by day 7.

In the initial in vitro cytotoxic studies on these compounds, a gradualreduction in the pH of the growth medium was observed as the cancercells metabolized over a seven day period. Therefore, it was believed tobe possible to adapt the prostate cancer cells to low pH between 6.5 and7.0. Hence, confluent PC3 cell lines were left incubated at 37° C. untilthe growth medium attained a low pH of 6.7-7.0. The cells were furtherpassaged twice into RPMI medium (pH 6.7-7.0) to allow them adapt to thislow pH environment. These cancer cells were then suspended in growthmedia buffered at pH 6.8 using KH₂PO₄, and plated in a 96 well plate.The cell suspension was made at a density of 2000 cells per 100 μL andthen plated in a 96 well plate at a density of 2000 cells/well. Cellsfrom the same passage were also plated in normal RPMI growth medium ofpH 7.4. The growth of PC3 cell lines under these two pH conditions wasmonitored over a 5-day incubation period.

MTT cell proliferation assay was done on each of day 1, day 2 and day 5.Growth of cells under these two conditions is comparable. Medium wasdrawn off the wells on day 0, day 3, day 5 and day 7 of incubation. pHwas measured and average pH calculated. They both gave a comparablegrowth curve as shown as FIG. 4.

Cells were incubated for 48 hours to give room for metabolism that mightlead to further lowering of pH before drug treatment. Cells were thentreated with six different concentrations of each drug (1 or 5) preparedin a growth medium of pH 6.8. Emetine dihydrochloride was used as apositive control. Due to cancer cell metabolism, the average pHgradually decreased further to as low as 6.4 by the fifth day of thisstudy.

In vitro activation of pH-responsive representative emetine prodrug 1and 5 was analyzed by measuring the change in ratio of cytotoxic IC₅₀values of each drug compared to that of emetine on day 5 in PC3 celllines under different pH conditions. As expected, the difference in thecytotoxicity of 1 and 5 compared to that of emetine reduced drastically(only about 2 fold difference from emetine) at pH<7.0. On the otherhand, there is more than 16 fold difference in the cytotoxicity of 1 and5 relative to emetine at pH 7.4, as shown in Table 6 below. Theincreased cytotoxicity of these prodrugs (1 and 5) at pH less than 7.0is thus in agreement with their activation to emetine under slightlyacidic environment. Hence, it appears that emetine is the majorcytotoxic agent when these compounds are subject to pH<7.0. In addition,the results at pH 7.4 compared to pH<7.0 indicate that these compoundsare far more activatable in the acidic cancer environment thusestablishing these compounds as potential prodrugs of emetine.

This became the in vitro model for performing a pH-responsive prodrugactivation assay.

TABLE 6 Compound IC₅₀ fold at pH 7.4^(a) IC₅₀ fold at pH <7.0^(a) 1 16.51.9 5 16.8 2.5 ^(a)IC₅₀ fold at each pH is determined as: IC₅₀ of eachdrug/IC₅₀ of emetine.

These results suggest that an appropriately designed emetine analogcould become a valuable cancer chemotherapy. The vital role of the N2′secondary amine is seen in the reduced cytotoxicity of all the N2′derived analogs. These analogs can be hydrolyzed to emetine at variablerates that depends on the substituent at the “tunable handle.” It isalso vital to note that these compounds are relatively stable at pH 7.4,indicating that emetine will most likely not be released in the blood orthe environment of normal tissue.

Example 15 In Vivo Toxicity Study

Each experiment or dosage involved three mice, and drug solutions weremade in 1% DMSO/PBS. Each experiment was done three times. A single doseof emetine at 100 MPK (milligrams per kilogram) killed all three micewithin 48 hours, while 33 and 11 MPK caused slight weight loss.

Two prodrug compounds were also tested. The tested compounds werelabeled “compound 1” and “compound 5” and are identified in the tablebelow.

Compound 1

Compound 5

The compounds were subjected to in vivo toxicity studies as follows.Compound 1 and Compound 5 in mice produced no noticeable toxicity at 100MPK or below (33 and 11 MPK). This observation was the same on the thirdand fourth day. On day 5, no noticeable toxicity effect was seen in micereceiving compound 1 and compound 5 at 11 and 33 MPK, but a slightweight loss was observed in those receiving these two drugs at 100 MPK.

While the invention has been described in terms of preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web in an ASCII text file labeled 8823-130777-US andis hereby incorporated by reference in its entirety.

1. A compound comprising formula (I):

where M is selected from the group consisting of:

and where:

X₁═H, —NO₂, —CO₂X₂, −OX₃, halogen, or C1-C4 alkyl, X₂ ═C1-C4 alkyl or H,X₃═C1-C4 alkyl or H, Q=OH, V+O—, V=metal ion, Y═H or C1-C6 alkyl, Z═H,C1-C4 alkyl, or halogen, p=0 to 8, and t=1 to
 4. 2. A compoundcomprising formula (I)

where M has the general formula:

and where V is a metal ion.
 3. The compound of claim 2, wherein V issodium or potassium.
 4. A compound comprising formula (I):

where M has the general formula:

where R8 is selected from the group consisting of:

where L=H₂N— and H₃CO— m=1-3. n=0-1, p=0 to 8, X₁═H, —NO₂, —CO₂X₂, —OX₃,halogen, or C1-C4 alkyl, X₂═C1-C4 alkyl or H, and X₃═C1-C4 alkyl or H.5. A compound comprising formula (I):

where M is not hydrogen and comprises a substituent effective to behydrolyzed by an enzyme preferentially expressed by cancer cells.
 6. Thecompound according to claim 5, wherein M comprises a peptide having thesequence His-Ser-Ser-Lys-Leu-Gln.
 7. The compound according to claim 5,wherein M comprises one of

where X is a protecting group or one or more amino acids.
 8. Thecompound according to claim 7, wherein X is selected from the groupconsisting of 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl,acetyl, and morpholino groups.
 9. (canceled)
 10. A method of deliveringemetine to a subject comprising administering to the subject aneffective amount of a compound according to claim
 1. 11-15. (canceled)